Project Summary This proposal seeks competitive renewal of a multi-PI project (Fu and Yeo), which aims to use global approaches to elucidate the regulatory principles of RNA binding proteins (RBPs) in mammalian genomes. Built upon our accomplishments in the past funding cycle, we propose to leverage the powerful experimental and computational tools we have developed to pursue four specific aims. In Aim 1, we will couple gain- and lost-of-function multi-target screens to deduce regulatory pathways at both splicing and polyadenylation levels. We will focus on determining the specific function of different RNA polymerase II (Pol II) subunits, rather than by the Pol II CTD alone, in the recruitment of RNA processing machineries for co-transcriptional RNA processing. In Aim 2, we will develop a general strategy for systematic identification of chromatin-associated RBPs to determine direct contribution of some RBPs to transcription and co-transcriptional RNA processing reactions. We will concentrate our efforts in dissecting a potential new pathway in epigenetic control of alternative splicing as well as broader roles of specific RBPs in direct transcriptional control. In Aim 3, we will use a novel strategy for identification and characterization of non-canonical RBPs. Focusing on a large number of newly identified finger zinc (Znf) proteins, we propose to determine their roles in binding to both DNA and RNA and deduce their transcriptome-wide interactions with RNA. We also propose to pursue a specific paradigm in this aim on a newly identified RBP known to associate with the nuclear pore to determine its role in selective mRNA nuclear export, which is pertinent to an ALS-regulated disease pathology. Combined, we believe that this comprehensive, interconnected, and hypothesis-driven research plan will greatly advance our understanding of regulated RNA processing and associated disease mechanisms.